Device and method for removing undesirable biological and/or chemical entities from biological fluids

ABSTRACT

A device removing a biological and/or chemical entity (C) from extracorporeal blood (B) is disclosed. The device has a hollow capture chamber with an inlet for the entry of the extracorporeal blood (B) and an outlet for the outflow of the extracorporeal blood (B) and a capture element inside the capture chamber having a reactant surface placed in contact with the extracorporeal blood (B) and a plurality of binding agents (A) for the biological and/or chemical entity to be removed (C) such that the biological and/or chemical entity (C), upon exiting the capture chamber, is removed from the extracorporeal blood (B) as linked to the reactant surface.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/096,466, filed Oct. 25, 2018, now U.S. Pat. No. 10,864,313,which is a 371 of PCT Application Ser. No. PCT/EP2017/059626, filed Apr.24, 2017, which claims the benefit from Italian Patent Application No.UA2016A002865, filed Apr. 26, 2016, the contents of each of which areincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a device for removing at least oneundesirable biological, biochemical and/or chemical entity, for exampletoxic substances and/or tumor cells, from a biological fluid volume, forexample blood, or rather blood temporarily taken from a patient by meansof an extracorporeal circuit [or extracorporeal circulation system(ECS)].

The present invention also relates to a corresponding extracorporealcirculation system comprising said device and a method for the removalof at least one undesirable biological and/or chemical entity from anextracorporeal blood volume.

In particular, the present invention relates to an “ex vivo liquidsurgery device” as well as an “ex-vivo liquid surgery” system and methodfor the removal of circulating tumor cells (CTCs) from the total bloodvolume (TBV) of a cancer patient, during a repeated procedure ofextracorporeal blood circulation, in order to decrease or avoidmetastasis spreading and risk of relapse.

STATE OF THE ART

Circulating tumor cells (CTC) are biological entities released into thecirculating bloodstream, mainly from the primary tumor, capable ofgiving rise to the so-called metastases (Zhank L. et al., Sci TranslMed, 2013; Baccelli I. et al., Nat Biotech, 2014; Aceto N, et al., Cell,2014). Since metastases are the leading cause of death caused by tumors,the biological and clinical significance of these cells isunquestionable. The capture and the removal of such cells from the bodyof a cancer patient could limit and/or prevent the risk of metastaticspread of the disease and possible relapses, as well as a conventionalsurgical approach of a solid tumor can limit the disease or even cure acancer patient.

CTCs were first identified in 1869, but only in the last 15 years anextensive scientific investigation has defined the role and thediagnostic, prognostic and predictive potential thereof. Their potentialclinical value as well as their clinical value in terms of prognosis areunquestionable. In fact, detecting, “capturing” and studying these cellsand the biomolecular characteristics thereof would allow for a moretargeted and efficient therapeutic treatment of the patient.Subsequently, removing all, or at least a large part, of said cells,from the bloodstream would allow for a drastically reduced or avoidedrisk of metastatic spread of the disease, thereby increasing thesurvival of patients or their disease-free survival. Until now, however,the only documented clinical use of CTCs is the prognostic one definedon the basis of their abundance. Moreover, up to now, the prognosticvalue of the number of CTCs in the peripheral blood has been documentedonly in breast, colon, and prostate cancers in the metastatic stage,demonstrating a better prognosis for patients who have less than 5CTCs/7.5 ml peripheral blood. All this is due to the intrinsiccharacteristics of the CTCs, i.e. their rareness, heterogeneity andplasticity.

In summary, CTCs have proved to be a valuable clinical tool only as aprognostic marker and not as a factor that can lead to a more targetedand effective therapy assisting in the choice of the drug(s) to be usedin order to increase the survival of patients (Joosse S A and Pantel K,Cancer Res, 2013; Alix-Panabieres C and Pantel K, Clin Chem, 2013; KrebsM G et al., Nat Rev Clin Oncol, 2014; Joosse S A et al., EMBO MolecularMedicine, 2014). Moreover, CTCs have never been a direct target of atherapeutic approach.

CTCs are rather rare cells, i.e. 1-10 cells per milliliter of peripheralblood and are often genetically, phenotypically and functionallyheterogeneous. These values are found in the majority of patients withsolid tumors in the metastatic stage (i.e. in all those where they wereanalyzed/studied). In solid tumors not in the metastatic stage theirfrequency is even lower. They may acquire stem-like properties andchange dynamically during time, converting from a more epithelial-likestate to one more mesenchymal-like and vice versa, through processesknown as Epithelial-to-Mesenchymal and Mesenchymal-to-EpithelialTransitions (EMT & MET). During these processes CTCs literally changetheir phenotype down-regulating specific antigens while up-regulatingothers. For example, during/through EMT, CTCs can lose the expression ofone key epithelial marker, i.e. EpCAM, and acquire a pivotal mesenchymalmarker, i.e. N-Cadherin, or a stemness-related marker, i.e. ABC-G2. CTCscan move inside the circulatory system singly or in clusters. Clusterspossess 23- to 50-fold increased metastatic potential in respect tosingle CTCs (S A Joosse et al., EMBO Mol Med, 2014; M G Krebs et al.,Nat Rev Clin Oncol, 2014; T Brabletz, Cancer Cell, 2012; Y Kang & KPantel, Cancer Cell, 2013; N Aceto et al., Cell, 2014). Hence, a numberof CTC populations exists, epithelial, mesenchymal, hybridepithelial-mesenchymal, stem-like, single CTCs, CTC-clusters. Allpatients with solid tumors are potential CTC carriers, regardless of thestage of the disease. Therefore, potentially all solid tumors,approximately 95-98% of all tumors, have CTCs. However, only some ofthem will show clinically relevant blood CTC dissemination. In fact,some kind of tumor disseminate only locally and/or not preferentiallythrough the blood vessels (e.g. head and neck cancer). Hence, onlyspecific kind of tumor could actually benefit from the removal of CTCsfrom the TBV. For example, tumors that can actually benefit from theCTCs removal will be those which metastasize more through the bloodvessels and that are more prone to disseminate CTCs far from the primarytumor also, such as breast and lung cancer. CTCs may also be present inthe early months of the disease. From this point of view, at theclinical level and for clinical utility, avoiding its dissemination assoon as possible can be far more efficient that inhibiting their spreadwhen the tumor has already metastasized in multiple sites. As mentionedabove, CTCs are mutually phenotypically heterogeneous, i.e. the antigensexpressed on their surface can be different from cell to cell, and theyare capable of mutating over time. This means that during theprogression of the disease they may acquire and/or lose antigens. Inother words, the circulating tumor cells constitute an uncommon cellpopulation consisting of several subpopulations defined as, for example,epithelial cells, hybrid epithelial-mesenchymal cells, mesenchymalcells, mesenchymal and/or epithelial circulating tumor stem cells;therefore, a set of biomolecular characteristics that make thenextremely difficult to detect and investigate.

Despite CTC importance, all known approaches, reported in prior art,refer to methods that delve with CTCs from just a single or few pointsof view. Moreover, CTCs have been investigated only as markers withoutdemonstrating their full potential. At present, no one exploit them astargets and without using any drugs that specifically target CTCs. CTCsare the “leukemic phase” of a solid tumor and hence they deserve thesame importance and a similar approach reserved to the primary ormetastatic tumor mass: an actual “liquid surgery”.

To date, the main systems for analyzing and capturing these cells arebased on their biological properties, for example by providing the useof individual antibodies directed against antigens expressed on theirsurface (e.g. in particular EpCAM), or physical properties, by beingcapable of selecting the CTCs by size or cell density, or oncombinations of biological and physical approaches. However, thesemethods exhibit the disadvantage of selecting cells that express onlyone or a few epithelial antigens and especially of analyzing only smallvolumes of peripheral blood, normally less than 30 ml.

Regardless of the type of method chosen, in fact, in most cases, theanalysis of the CTCs is always carried out on a small volume ofperipheral blood, e.g. 5-10 milliliters. If on the one hand a smallwithdrawal of blood is cheap and poorly invasive for patients, on theother hand it considerably decreases the chances of detecting cells thatare so rare. As demonstrated by Coumans et al. Clinl, Cancer Res, 2012,as the volume of analyzed blood increases, the likelihood of detectingand possibly removing the CTCs also increases. In addition, it has beendemonstrated (through a mathematical extrapolation) that in 99% ofpatients with a metastatic tumor at least one CTC can be detected oncethe entire volume of blood has been analyzed, i.e. the total bloodvolume (TBV). The TBV is the volume of blood that usually circulate in ahuman being/patient. Normally this volume consists in up to about 3-5liters. It follows that to detect and then delete the greatest number ofcirculating tumor cells it is necessary to analyze a volume of bloodthat is as large as possible.

To then perform a true liquid biopsy/surgery and therefore detect andremove the greatest possible number of CTCs, a substantial increase inthe volume of peripheral blood that is analyzed, and therefore in theyield of the CTCs themselves, is required. Since it is obviouslyimpossible to collect large quantities of blood from a patient withoutcausing damage to him/her, one possibility is to use a method ofextraction of the CTCs connected to an extracorporeal circulation system(ECS). As stated by Allard W J and Terstappen L W, Clin Cancer Res,2015, an ECS-based system would allow for increasing the volume ofanalyzed blood up to at least 1 “total blood volume” (TBV), i.e. about3/5 liters in total, if not even up to 4/6 TBVs, and thus for increasingthe number of circulating tumor cells that are potentially detectableand removable.

Although devices for capturing CTCs by means of extracorporealcirculation systems and size exclusion filters suitable for the captureof tumor cells are known, these devices have the disadvantage of beingable to analyze blood volumes equal to a maximum of 1 to 3 TBV and ofnot detecting and capturing cells that are smaller than the pores of thefilter independently of the antigens expressed by the CTCs, thusobtaining only a limited yield of capture. In particular, the knowndevices often do not capture CTCs that express antigens other thanepithelial ones. Also, these devices are stationary, motionless filtersor tubes that are clearly prone to be obstructed or clogged up if manyliters of blood and different cellular elements pass through them. Forexample, known devices are mainly targeted to just one population ofCTCs, e.g. epithelial, and were generally based on micro-fabricatedfilters included in an apheretic extracorporeal circulation. It is clearthat a still too small volume of blood can be screened with such devicesdue to the nature of the apheretic procedure (designed to screen alimited number of liters of blood) and because after a number ofcycles/milliliters of blood the system will be inevitably clogged orsaturated, probably inducing dangerous blood clotting also. Moreover,such filters certainly require a low blood flow speed to try to avoidearly clogging and saturation.

It is an object of the present invention to overcome the above mentioneddrawbacks of the known systems for detecting and capturing CTCbiological and/or chemical entities and to provide a device and a methodwhich are more efficient and functional.

DESCRIPTION OF THE INVENTION

A device, a system and a method for the removal of at least oneundesirable biological and/or chemical entity from an extracorporealblood volume are provided according to the independent claims.

The device according to the present invention comprises a hollow capturechamber having an inlet for the entry of blood from an extracorporealsystem/circuit and an outlet for the outflow of the extracorporeal bloodand the re-entry into the normal body circulation. The capture chamberinternally comprises at least a capture element movable relative to thecapture chamber and having a reactant surface which is placed in contactwith the extracorporeal blood. The reactant surface comprises aplurality of binding agents for the biological and/or chemical entity tobe removed such that at the outlet of the capture chamber the biologicalentity is removed from the extracorporeal blood, the biological and/orchemical entity being bonded to the reactant surface. In particular, thetotal volume of extracorporeal blood flowing inside the capture chamberis comprised between 1 and 6 TBVs and wherein each TBV consists of up to5 liters of blood.

This device is practical and effective in its operation.

By employing the device according to the present invention, it ispossible to realize an “ex-vivo liquid surgery” for removing biologicalentities (for example CTCs) from the blood of a patient thereby limitingor avoiding tumor progression, that is similar to a conventionalsurgical approach of a solid tumor that can limit the disease or evencure a cancer patient.

According to the present invention, it is possible to capture thegreatest possible number of undesirable biological and/or chemicalentities during their circulation in the bloodstream. Moreover, thepresent invention allows for analyzing a high volume of blood, even upto 300 ml/minute, a value that is much higher than any blood sampling.In fact, known systems for the analysis of CTCs, based on simple bloodsampling, analyze a maximum of 1-30 ml of peripheral blood. Moreover,given the rarity of the CTCs, the chance of detecting “false negative”cases is statistically very high. Other systems can instead analyze ahigher amount of blood but never reaching the volume indicated by theapproach according to the present invention, most importantly, neverusing a device implying a dynamically driven cell-to-surface contact.Through an in vivo system for capturing CTCs, for example, said CTCs canbe directly captured in a patient's vein without collecting blood fromsaid patient. However, a system of this kind, although screening thepatient's blood for about 30 minutes, can analyze no more than about1.0/1.5 l of blood and capture the CTCs thanks to a single antibody thatonly binds the EpCAM molecule, if expressed on the CTCs.

Instead, the device and the system according to the present inventioncan screen from 3 to 5 liters of blood (=1 Total Blood Volume [TBV]) upto about 30 liters of blood, i.e. 4/6 TBVs, practically identical to oreven greater than what is obtained with a traditional ECS system, forexample, leukapheresis or hemodialysis for renal disease or failure.

In a particular embodiment of the present invention, to increase thepossibility to catch all the CTCs, the TBV is examined more than onetime, up to at least about 14 times (14 TBVs), that is to say up toabout 72-74 liters of blood. In this way, the device of the presentinvention allows the capture of CTCs from up to about 14 TBVs of acancer patient in order to allow the recovery of a highly significantamount of CTCs. The recovery of a highly significant number of CTCs willallow statistically significant clinical information (after downstreamcellular and molecular analysis), allowing critical clinical decisionsand personalized medicine approaches.

The removal of CTC sub-populations, epithelial, mesenchymal and/orhybrid, singly or in clusters, is obtained by the combination ofmolecular (antigen expression dependent) and physical (size dependent)approaches. The device according to the present invention can comprise anumber of different binding reagents targeted towards CTC-specificantigens. In particular it comprises EpCAM, E-Cadherin, N-Cadherin, CD44and its isoforms (e.g. CD44v6 and CD44v8), selectins, ABC-proteins,MUC1, FGFRIIIc, and other “every kind of target/antigens” related toepithelial, mesenchymal and cancer stem-like phenotypes. At least, 5 to6 binding molecule type are necessary to enrich and grab most of the CTCpopulations. Preliminary data, confirmed that the selection of thepreviously reported CTC-specific antigens herein presented is optimal.With this combinatorial approach, in fact, epithelial, mesenchymaland/or hybrid CTCs have been detected in cell line models during EMT andin particular in cancer patients during progression also. These resultsclearly demonstrate the efficiency of the device and the methodaccording to the present invention.

In fact, thanks to the use of a plurality of binding agents (at least 6antibodies) distributed on the reactant surface, a greater number ofentities to be removed can be intercepted, as they are directed to agreater number of antigens expressed by such entities. Hence, all theCTC sub-populations, epithelial, mesenchymal and/or hybrid, singly or incluster are targeted and captured by the device. Specifically, thedevice serves as a trap for the circulating entities to be removed,which by interacting with the plurality of binding agents on thereactant surface of the capture element, remain bound to this surfaceand do not continue along the bloodstream's path of the blood flow. Theblood exiting the device will therefore be devoid of the undesirableentities. At a later time, these removed entities can be collected fromthe surface of the capture element and analyzed downstream to supportclinical decisions through clinically relevant data related to theirnature. Hence, in its various embodiments, the device according to thepresent application is aimed at treating cancer, removing them fromblood, and enriching CTC allowing further downstream analysis. In turn,these analyses allow a significant monitoring of cancer features, duringprogression also, giving to the clinician a statistically significantnumber of cancer cells to be studied.

The shape and the size of the capture chamber and capture element aresuch as to allow for a flow of blood therein up to about 200-300ml/minute and to ensure the largest possible contact surface between thefilter element and the blood flow.

For example, two types of devices and thus of capture chambers areconceivable: a microscopic one and a macroscopic one.

The microscopic device can provide a minimum length of 2.0 cm and aminimum diameter of about 1.2 cm, while the macroscopic device canprovide a minimum length of between 4.0 and 5.0 cm and a minimumdiameter of about 1.2 cm. Of course, different dimensions areconceivable on the basis of appropriate hemodynamic studies.

The feature according to which the capture element is movable relativeto the capture chamber allows capturing the undesirable biologicalentities (i.e. CTCs) and helping these entities to get as close aspossible to the reactant surface of the device (able to catch/grab theCTCs). In fact, due to the mobility of the capture element, the reactantsurface is better in contact with the blood flowing during theextracorporeal phase of an extracorporeal circulation procedure.Moreover, the moving structure can be headed and/or followed, in thedirection of the blood flow, by a filter structure.

The movement of the capture element, that represents the core structureof the device, can be guided by the blood flow or by an external force.The movement, in turn, will lead to a stirring of the blood and to acirculating path in the chamber increasing the contact of cells with thereactant surface and the internal wall of the capture chamber. Thismovable element also facilitates the fluid circulation, making easier tocapture numerous TBV, without the risk of system clogging or bloodclotting and damage.

The undesirable biological and/or chemical entity to be removed can beany cell, undesirable entity or pathological biochemical structurenoxious for the human body, which can be detected within the blood andhas specific recognizable traits. Specifically, this element can be acirculating tumor cell (CTC). In this way, through the device accordingto the present invention, the spread of the CTCs in a patient's body,and consequently the development and growth of metastases, can beprevented, or at least greatly reduced. The device can potentiallytarget all solid tumors, i.e. more than 95% of tumors. However, asalready mentioned, some types of tumors are more prone to distantdissemination through blood vessels and hence they are more biologicallyand clinically suitable for the use of the device according to thepresent invention in its various embodiments.

Summarizing the above, by using the device according to the presentinvention. it is possible to combine for the first time the examinationof up to 6 TBVS or up to 14 TBVs with three different and complementaryapproaches: a molecular, a physical and an engineering-based fluiddynamic approach, the last of them being a first in art method tocapture CTCs. This combination approach ensures a) a higher level ofcapture efficiency, providing the removal of epithelial, mesenchymal,hybrid epithelial-mesenchymal, and stem-like CTCs, singly and/or inclusters, b) a sustained and reasonably fast blood flow and c) no riskof system clogging or blood clotting and damage.

The binding agents are distributed on the surface of the capture elementin order to capture the undesirable elements within the bloodstream bybinding to antigens, proteins or receptors on the surface of theundesirable entity to be removed. Specifically, the binding agents maycomprise one or more antibodies, adhesion proteins, aptamers,oligo-aptamers, or other organic molecules specifically directed againstany undesirable compound or element that must be removed from thebloodstream. In the case of CTCs, these are captured by the bindingagents due to the presence of antigenic epitopes on the membrane of thetumor cell, which depend on the subpopulations of the target cells, forexample, epithelial cells, hybrid epithelial-mesenchymal cells,mesenchymal cells, mesenchymal and/or epithelial circulating tumor stemcells (singly or in clusters) and/or any other CTC subpopulation thatcan be detected in the blood of cancer patients. The collection ofdifferent subpopulations of cells can be obtained thanks to suitable andcustomizable mixtures of antibodies and/or molecules recognized asbinders for the cells of interest. These mixture of capturing moleculescan be different from cancer to cancer, from patients to patients,depending on the antigens present on the CTCs. The discovery andselection of the more suitable antigens with which functionalize thesurfaces of the device can depend on the discovery or investigation ofspecific antigens on the primary/metastatic tumor tissue or utilizing aliquid biopsy approach (e.g., Cell Search, DEPArray, ISET, etc., . . .). To increase the likelihood of capture, binding agents or antibodiesdirected against alternative antigens/phenotypes expressed by the CTCcells to be removed may be used, i.e. in addition to those traditionallyrecognized, as well as those identified more recently and characterizingthe CTCs deemed more aggressive (Barriere G et al. Ann Transl Med 2014).

To optimize the capture of the entities to be removed, the binding ofthe antibodies and/or other biological biomolecules or binding agentsmust be such that their antigen-binding moiety (e.g. Fab for antibodies)is positioned with an orientation that goes from the surface of thecapture element in the direction of the inner surface of the capturechamber. In this way, the likelihood of binding to the analyte (forexample the CTCs) can be maximized.

The capture of undesirable entities within the bloodstream can beachieved by means of specific techniques, such as controlledimmobilization (Qian W et al., Clin Chem. 2000; Jung Y et al., AnalBiochem. 2008; Kumada Y, Biochim Biophys Acta 2014; Crivianu-Gaita V andThompson M, Biosens Bioelectron 2015).

Advantageously, Fab fragments of an antibody alone may be immobilized.Compared to the more traditional technique of immobilization of thewhole antibody, the binding of Fab fragments proves to be able to reachhigher surface densities, thereby obtaining a higher binding capacityfor the analyte (Crivianu-Gaita V and Thompson M Biosens Bioelectron2015).

The type of binding of the antibody/Fab to the surface can be determinedon the basis of the material chosen for the reactant surface of thecapture element. The immobilization of antibodies/Fab fragments can be,for example, carried out on gold (Au)-coated, silicon (Si)-based, andpolysaccharide-based surfaces, or on plastic and generally inorganic,yet always biocompatible, surfaces like polyurethane, polypropyleneand/or polycarbonate.

The possibility to functionalize the device with different bindingagents, i.e. antibodies, depending on the disease, is of particularinterest. In fact, it is conceivable that a first group of antibodies ispotentially present on each device and directed against antigensexpressed by a subpopulation consisting of CTCs, while a second groupcan be selected on the basis of the disease under examination.

Specifically, by way of example, EpCam (CD326), E-Cadherin (CD324) andEGFR (epitheliality), N-cadherin (CD325) and OB-cadherin (cadherin 11)(mesenchymality) can be considered as “standard” antigens, and at least,for example, CD44v6 (colon cancer; tumor stemness), CD44v8 (breastcancer; tumor stemness), Her-2 (breast cancer) and ABC-G2 (tumorstemness) as “disease-specific” antigens.

It should be noted that both the different binding agents and the shapeof the device itself, and in particular the shape of the reactantsurface as well as the mobility of the capture element relative to thecapture chamber, affect the action of capture of the entities to beremoved, for example the CTCs. In fact, the binding agents detect andcapture various subpopulations of entities to be removed, while theshape and the movement of the reactant surface allow for increasing thecontact surface as well as the contact probability between the captureelement and the blood of the patient, thereby allowing for a greaterlikelihood of binding between the entities and the different bindingagents.

The capture chamber of the device according to the present invention ishollow and must have a shape that allows the blood flow to circulateinside it in a fluid and smooth way. In one embodiment of the invention,the capture chamber has a cylindrical shape and the central movablecapture element extends longitudinally within the capture chamber. Boththe capture chamber and the capture element can be entirely made up ofan alloy or material biocompatible with a coating such as to prevent apossible interference with coagulation, normal blood cells or otherphysiological processes. In order to promote adhesion of the undesirablebiological and/or chemical entities onto the reactant surface of thecapture element, said surface can be advantageously coated with a thinlayer of gold or other biocompatible material. In addition, in order notto impair the blood flow, the reactant surface of the capture elementmay be made of a deformable material.

In one embodiment of the invention, the movable capture element canrotate about a longitudinal axis of the capture chamber. This allows formaximizing the contact surface between the reactant surface of thecapture element and the blood flow containing the undesirable entitiesto be removed. Furthermore, the rotation of the capture element allowsthe blood to flow away better, thereby avoiding system clogging and/orblood damage and clotting. In fact, the continuous movement can help tomaintain the fluidity of the blood and prevent the formation of clots.The movement of the central element can also be allowed by the presenceof four, preferably small, magnets, two positioned at its ends and twoat the ends of the capture chamber. The magnets thus positioned willmaintain the central element in levitation, thereby reducing thepossible friction that could be created between the central element andthe outer chamber.

An anticoagulant, such as heparin, can also be added in order to preventunwanted coagulation phenomena. The movement and the anticoagulant havethe purpose of maintaining the blood characteristics intact, thusallowing for the maintenance of the clinical safety of the patient.

To further increase the safety of the patient, the blood pressure can becontrolled both in the input and output of the capture chamber such thatit remains even.

The rotation of the capture element relative to the capture chamberaround a longitudinal axis can take place either by the mere effect ofthe blood flow, and therefore without the application of a dedicatedexternal force, or by application of an externally regulated, continuousdriving force.

The rotation of the capture element is pivotal to allow a better capturecompetence of the overall device in respect to an apparatus withoutrotating elements. This data depends on the capability of the rotatingcapture element to help the target CTCs to get in proximity with thereactant surface of the device.

In order to allow the undesirable entities captured by the reactantsurface to be analyzed, the capture element can be removed from thecapture chamber. This may be accomplished by means of acoupling/uncoupling system located at the entrance and at the exit ofthe capture chamber.

Moreover, the binding agents are linked to the surface of the deviceutilizing biochemical linkers able to be cleaved only when the procedurehas been stopped and the device taken outside from the EC. This detailallows the recovery of cells from the device in order to study them andobtain clinically useful information with cellular and molecular methodsalready known.

In one embodiment of the invention, the reactant surface of the captureelement comprises a helical structure. In particular, the helical orscrew or Archimedes' screw structure extends longitudinally around arotation axis and is secured to the capture chamber by means of afastening system. More specifically, the movable capture element isshaped like an helix or spiral. This particular shape is used to ensurean even flow of the blood fluid and at the same time a greater surfacethat is in contact with the circulating blood so as to increase thelikelihood of binding between the entity to be removed, for example thetumor cell, and the binding agents, for example the antibodies.Preferentially, the helical surface may extend along the entire lengthof the capture chamber. However, a configuration in which the length ofthe helix is less than the length of the capture chamber and insidewhich there is a plurality of helical structures placed in series mayalso be used. Of course, through such a configuration, the length of theentire device must be necessarily increased.

According to the embodiments of the present inventions, the movablecapture element is configure to facilitate the contact between thereactant surface of the device and the target cells. Hence, every kindof moving helix, double helix, or other moving 3D structure made up tofacilitate this contact is included in this invention.

According to the microscopic type mentioned above, the device may, inparticular, have a minimum diameter of between 0.5 and 0.8 cm per singlehelix, in which the helix can perform 5 rotations around its centralaxis, i.e. approximately one each 0.4 cm.

According to the macroscopic type mentioned above, the device may have aminimum diameter of between 0.5 and 0.8 cm per single helix, in whichthe helix can perform 5 rotations around its central axis, i.e.approximately one each 1.0 cm.

In particular, the movable capture element of the device, first in artin the field of CTC study, is a rotating system with the aim of drivingcells as near as possible to the reactant surface and of that ofinternal wall of the capture chamber. In an embodiment of thisinvention, the capture element is similar to an Archimedes screw,including all different shapes, dimensions and types of helices orsimilar 3D moving structures and appropriate capture chambers or housingtubes. The rotational speed is controlled by the blood flow through thechamber depending from a CEC system. The flow speed can be adjusted inorder to throw/cast blood cells and CTCs towards/on the surface on thesame moving capture element and/or on the surface of the internal wallof the capture chamber. At this stage of the procedure, cells hit thereactant surface of the helix. Cells can be captured at this point ordriven by the thrust of the moving capture element itself towards theinternal wall of the chamber. In this system, several rounds of totalblood can be screened to obtain a maximum capture efficacy as alreadymentioned. A device having a rotating capture element helps therefore toobtain a maximum capture effectiveness.

In another embodiment of the invention, the device further comprises oneor several conical structures consisting of thin cables made ofbiocompatible material. These structures have a filtering mesh surfacemade of holes (pores) with a diameter greater than 100 μm. The conicalstructure can be positioned upstream or downstream the capture elementand in line with this element. The conical structure may be similar, forexample, to a spider filter for the protection from distal and vascularemboli. In particular, the capture element can be configured so as tocomprise a single conical structure that is fixed or can rotate around alongitudinal axis passing through the tip of the cone, in which the baseof the cone receives the input blood flow. However, to improve thefiltering effect, the capture element may be configured so as tocomprise a plurality of conical structures positioned in series alongthe direction of the blood flow.

This conical web filter is technically designed to block very large CTCsbut in particular tumor micro emboli or CTC-clusters. These elementspossess higher metastatic potential with respect to single CTCs. Hence,the “spider filter” like filters plays a pivotal role in preventing thedissemination of blood micro emboli, limiting metastatic relapse andincreasing device safety.

The pore diameter has been selected to block CTC clusters, but largeenough to avoid risk of system clogging or blood clotting and damage.The selection of a diameter of about 60 to 100 micrometers has a furtherjustification. CTC-clusters are born from oligo-clonal tumor cellgroupings and not from intravascular aggregation event (Aceto N. et al.,Cell, 2014). Hence, accepting a cell cluster is composed of 2 or morecells (range from 2 to 19, average 10.5, as reported by Sarioglu A F etal., Nat Methods, 2015) and assuming that single CTC diameter rangesfrom 8 to 16 micrometers, 12 in average (Hosokawa M et a., PlosOne,2013; Patrizia Paterlini-Bréchot, Cancer Microenviron 2014; Vona G etal., Am J Pathol. 2000), it can be inferred that an average cluster ofabout 10 cells presents a dimension around 120 micrometers (range from96 to 160).

In a further embodiment of the invention, the device may comprise acombination of a capture element having a helical structure with one ormore conical structures. This allows for further enhancing theperformance of capturing the entities to be removed as well as theclinical safety of the device since it may block potential clots andemboli. In particular, such a device may be configured so that thehelical structure precedes the conical one, referring to the directionof the blood flow.

One embodiment of the invention can provide for the presence of multiplecomplete devices (capture chamber and central capture element)positioned in series like the pearls of a necklace.

The core structure of the device, that can be the helix element combinedwith a web-like filter, can be coated with molecules targeted towardsCTC-specific antigens in order to specifically catch them during theirflow through the device.

Of course, alternative forms to the helical or conical ones as regardsthe reactant surface of the capture element are possible and fall withinthe invention. The important thing is that the shape is such as toensure, on the one hand, a good surface of contact with the blood flow,and on the other, a fluid blood flow without the risk of clots, embolior block system.

Advantageously, the plurality of binding agents is distributed evenlyover the entire reactant surface of the movable capture element. Thisallows the capturing action to be distributed over the entire reactantsurface of the capture element.

Alternatively, the binding agents can be concentrated in specific areasof the reactant surface of the movable capture element. This isparticularly advantageous when using binding agents of a differentnature. In other words, a “progressive” distribution of the bindingagents on the reactant surface is possible. This means that the bindingagents, for example the antibodies, directed against certain antigenscan be placed only at one end of the reactant surface, while theremaining may adhere to other adjacent regions. This configuration,other than allowing for an efficient capture capacity, allows theentities to be detected in a more simple way. In fact, once the captureelement has been removed, the entities can be easily identified byanalyzing the different regions of the reactant surface that arespecific for different subpopulations of entities to be removed.

In a further embodiment of the invention the binding agents aredistributed evenly or only concentrated in specific areas on the innersurface of the capture chamber. In this way, it is possible to increasethe capture action of the device. More specifically, the distribution ofthe binding agents on the internal surface of the capture chamber aswell as on the reactant surface of the capture element allows that ahigher quantity of blood is treated by the device.

The moving capture element, the conical filter and the internal wall ofthe capture chamber can all have reactant surfaces. These surfaces arecoated with multiple binding agents directed towards different CTCantigens and consequently towards different CTC populations. Accordingto this configuration, the total volume of blood exiting from thecapture chamber, passed through the device, is cleaned of CTCs, the CTCsbeing captured by the internal reactant surfaces of the device. Byrepeating this approach a number of times (6 up to 14 times), for up toabout 4 to 5 hours at a blood flow speed up to 300 ml/min, it ispossible to screen up to 14 TBV per treatment with the aim to removemore than 90/95% of CTCs from the TBV.

As already mentioned, the various embodiments of the present inventioncan comprise one or more capture chambers (housing tubes plus theinternal moving elements plus the conical “spider” filters) organized insequence. Each single capture chamber is coated, on its internalsurfaces, with one or more types of binding molecule (e.g. antibodies oraptamers) against all the known CTC sub-population (epithelial,mesenchymal and/or hybrid, singly or in cluster). In another embodimentof the present invention, at least 3 or 4 chambers are foreseen. Thedifferent chambers are organized in sequence following the direction ofthe blood flow. Each chamber is coated with binding molecules against aspecific type of CTC sub-population (e.g., the first chamber againstmesenchymal CTCs, the following chamber against epithelial CTCs and thelast chamber against cancer stem-like cells).

It is noted that the possibility to analyze up to 6 TBV or up to 14 TBVof patient's blood is guaranteed by the fact that the capture elementcan move, i.e. can rotate around its longitudinal axis in blood flowdirection, and/or by the fact that conical structures are present actingas filters for clusters of cells and/or by the fact that the bindingagents are not only present on the surface of the capture element butalso on the internal surface of the capture chamber and/or on thesurface of the conical structures.

The system for the removal of biological and/or chemical entitiesaccording to the present invention is based on the integration of theremoval device mentioned above into an extracorporeal circulationsystem, similar to therapeutic leukapheresis or hemodialysis.

In particular, the system comprises extraction means for extractingvenous blood from a patient's vascular system and input means forintroducing the extracted blood into the removal device. In order toextract the filtered blood from the device and complete the cycle, thesystem further comprises output means and re-entry means forreintroducing the blood extracted from the device—and thus filtered—intothe patient's vascular system.

Moreover, to prevent the formation of clots and to keep the temperatureof the blood flow under control, the system comprises a means forintroducing an anticoagulant upstream of the removal device and atemperature control unit downstream of said device.

This system is capable of capturing and then removing any undesirablebiological and/or chemical or biochemical entity, such as for examplethe CTCs, from the blood of a patient along the path travelled by theseentities within the blood circulatory system. When the entities passthrough the removal device, said entities (for example CTCs) areretained by the binding agents (for example the antibodies) placed onthe reactant surface of the capture element. Other types of entities orblood cells, instead, may continue their path within the circulatorysystem. The passage of the blood to be analyzed and filtered through theremoval device takes place for a predetermined value of blood volume,after which the extracorporeal circulation system is removed, as well asthe device. The latter can be opened and the entities can be directlyexamined on the reactant surface of the capture element, or removed fromit for further molecular assessments.

Advantageously, once functionalized with the selected binding agents,the device can be assessed, i.e. tested, through the provision of an invitro flow apparatus that mimics the circulation of a known volume ofblood ex vivo or of any synthetic substance behaving like blood. Thisapparatus will enable for investigating the interaction of entities tobe removed, for example “model” tumor cells, with the functionalizeddevice and for simulating the in vivo venous blood flow conditions invitro. The flow can be sustained by a peristaltic pump to allow theblood to repeatedly interact with the functionalized device. Such anapparatus entails the use of a blood sample (taken from a healthy donor)added with entities to be removed, such as for example tumor cells fromestablished tumor cell cultures with characteristics similar to those ofthe CTCs, epithelial, mesenchymal, hybrid epithelial-mesenchymal, andstem-like CTCs, singly and/or in clusters. To prevent mechanical damageto the cells, the blood must not pass through the peristaltic pump.Specifically, the blood of the healthy donor—no more than 300/500 ml—ispassed several times through the ex vivo apparatus and the device untilreaching a TBV that is reasonably similar to what could be achieved in apatient. This apparatus will also be used to understand which captureelement or combination of capture elements in terms of shape,arrangement and quantity, has the best abilities of selecting andcapturing the entities to be removed (CTCs), while maintaining thenormal characteristics and fluidity of the blood. For example, such anapparatus will be extremely important to understand if the deviceinduces formation of clots or cell clusters which might affect theclinical safety of the device.

The ex-vivo method for removing at least one biological and/or chemicalentity from a volume of extracorporeal blood of a patient according tothe present invention comprises the steps of introducing theextracorporeal blood into a hollow capture chamber internally comprisingat least one capture element, wherein the capture element is movablerelative to the capture chamber, filtering the extracorporeal blood bycontact with a reactant surface of the movable capture elementcomprising a plurality of binding agents for the biological and/orchemical entity to be removed, and extracting the extracorporeal blooddevoid of said biological and/or chemical entity from the capturechamber.

In particular, the total volume of extracorporeal blood (B) flowinginside the capture chamber (10) is comprised between 1 and 6 TBVs andwherein each TBV consists of up to 5 liters of blood.

To increase the possibility to catch all the CTCs, the TBV is examinedmore than one time. In particular, according to the present inventionthe TBV is screened more than one time, up to at least about 14 times(14 TBVs), that is to say up to about 72-74 liters of blood. In thisway, the method of the present invention allows the capture of CTCs fromup to about 14 TBVs of a cancer patient in order to allow the recoveryof a highly significant amount of CTCs. The recovery of a highlysignificant number of CTCs will allow statistically significant clinicalinformation (after downstream cellular and molecular analysis), allowingcritical clinical decisions and personalized medicine approaches.

The method further comprises the step of stopping the introduction ofextracorporeal blood into the capture chamber after introducing apredetermined volume of blood, and removing the capture element toanalyze the nature and quantity of the biological and/or chemical entityremoved and present on the reactant surface.

The method therefore comprises passing the blood of a patient through adevice or system described above, simultaneously contacting the bloodwith the reactant surface of the capture element of the device,consequently contacting it with the selected appropriate binding agentsor antibodies. The step of adhesion of the binding agents to theentities to be removed can occur by means of various controlledimmobilization techniques.

Therefore, the method successively comprises the step of temporaryremoving the venous blood from a patient's vascular system through anextracorporeal circulation system, the step of adding heparin (or othersimilar anti-coagulant) to prevent unwanted coagulation phenomena, thestep of introducing the blood into the device, thereby contacting theblood and hence the entities to be removed (for example CTCs) present inthe blood of the patient with the selected binding agents for apredetermined period of time, the step of adding an effective amount ofi.e. protamine to reverse the action of heparin when the treatment isfinished and the final step of re-introducing the blood into thepatient's vascular system.

The actual treatment time according to the method of the presentinvention can vary but depends approximately on the time it takes 4 or 6TBVs to pass through the device or up to 14 TBVs. In particular, themethod provides for analysis and capture of up to 5 liters or up to72-74 liters of blood, i.e. approximately 300 ml/min. The treatment timeof such a quantity of blood can therefore approximately vary between20/30 minutes and 3 to 4 hours.

It should be noted that the device, and hence the system according tothe present invention, can be used both in a phase that is immediatelysuccessive the diagnosis and also in the monitoring of the progressionof the disease and of the response to possible therapies. In themetastatic and/or terminal phase, the device, may increase the time toprogression, progression free survival and/or improve the quality oflife of patients with tumors. The device can also be used as a“curative” device thanks to its properties of removal of undesirableentities, such as the CTCs responsible for the metastatic spread.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects of the present invention will become moreapparent in the light of the following description of a few preferredembodiments described below.

FIGS. 1 a and 1 b are schematic representations of the device accordingto the present invention in a longitudinal view (a) and in cross section(b);

FIG. 2 is a schematic representation of a capture element according toan embodiment of the present invention;

FIG. 3 is a schematic representation of the capture element of FIG. 2with a progressive distribution of the binding agents on the reactantsurface;

FIGS. 4 a, 4 b, 4 c and 4 d are schematic representations of a captureelement according to a second (a, b) and a third (c, d) embodiment ofthe present invention in a longitudinal view (a, c) and in cross section(b, d);

FIG. 5 is a representation of an extracorporeal circulation systemaccording to the present invention;

FIG. 6 shows a detail of the extracorporeal circulation system accordingto an embodiment of the present invention;

FIG. 7 shows a detail of the extracorporeal circulation system accordingto another embodiment of the present invention; and

FIG. 8 schematically shows a flow diagram of a method according to thepresent invention.

FIG. 1 is a schematic representation of the device 1 for the removal ofat least one undesirable biological and/or chemical element C (not shownin the figure) from an extracorporeal blood volume B. In particular, thedevice is suitable for the removal of circulating tumor cells. Thedevice 1 essentially consists of a capture chamber 10 having an inlet 12for the entry of the extracorporeal blood B and an outlet 14 for theoutflow of the blood B. As can be seen from the comparison of FIGS. 1 aand 1 b , the capture chamber has a hollow cylindrical shape and theblood B flows through the cavity of circular cross-section.

Inside the capture chamber 10 there is the capture element 20; 20′; 20″,which, thanks to the presence of a reactant surface (not shown in thefigure) placed in contact with the blood B, is able to capture andremove the undesirable biological and/or chemical entity C from thebloodstream B.

FIG. 2 shows an embodiment of a capture element 20 to be placed insidethe capture chamber 10. This consists of a longitudinal element 24parallel to the longitudinal axis a of the capture chamber 10 aroundwhich a helical-shaped reactant surface 22 is wound. Specifically, thereactant surface 22 has the shape of an Archimedes' screw. The bindingagents A (represented in the figure by arrows) are distributed on thereactant surface 22, the which agents are used to capture the biologicaland/or chemical entity C to be removed, represented by a circulatingtumor cell. As can be seen in the figure, the capture element 20 canrotate around the longitudinal axis a of the capture chamber 10. Thisrotation is caused by the blood flow B which drives the reactant surface22 into a helical motion. In this way, each part of the reactant surface22 can come into contact with the blood flow B.

FIG. 3 shows the particular case in which the binding agents aredistributed in a circumscribed manner in certain regions of the reactantsurface 22 of the capture element 20. In particular, the figure showssix different rectangular regions defined on the helical-shaped reactantsurface 22, which in FIG. 3 is arranged on a single plane for reasons ofclarity. Each of the six regions displays a binding agent A of adifferent nature (A₁, A₂, A₃, A₄, A₅ and A₆), so that differentsubpopulations of tumor cells C (in this case six) associated withdifferent antigens may be simultaneously captured and localized on thereactant surface 22 in an easier and faster way. Alternatively, thedifferent types of binding agents can be adhered to the central devicewith an even concentration but not located on specific surfaces, i.e. bydistributing them homogeneously over the entire surface of the device oron the internal wall of the capture chamber.

FIG. 4 shows another embodiment of a capture element 20′; 20″ to beplaced inside the capture chamber 10. According to this embodiment, thecapture element 20′; 20″ has a conical structure and is positionedinside the capture chamber 10 so that the blood flow enters through thebase of the conical structure. The capture element may comprise a singleconical structure 20′ (FIGS. 4 a and 4 b ) or a plurality of conicalstructures 20″. Specifically, FIGS. 4 c and 4 d show the case in whichtwo conical structures are placed in series in the direction of theblood flow B. The conical structure 20′; 20″ of this embodiment, inparticular, consists of thin cables made of biocompatible material ontowhich the binding agents A adhere. The conical structure 20′; 20″ has acircular base and extends symmetrically around a longitudinal element24′; 24″ parallel to the longitudinal axis a of the capture chamber 10.

FIG. 5 shows an extracorporeal circulation system 100 according to thepresent invention. Through a system of cannulae 2 the blood B can beextracted from the vascular circuit of a patient P and through anothersystem of cannulae 3 it can be introduced into the removal device 1. Ameans 6 for the introduction of an anticoagulant is inserted between thetwo systems of cannulae 2 and 3. After passing inside the device 1, theblood B can be extracted through suitable dedicated means 4, and bypassing through a temperature control unit 7, reintroduced through a newsystem of cannulae 5 into the body of the patient P. An appropriatemeans 8 for the reintegration of fluids, for example for neutralizingthe effect of the anticoagulant, is inserted in the vicinity of thesystem of cannulae 5 for reintroducing the blood B.

FIG. 6 shows a detail of the system 100 with regard to the inlet 12 andthe outlet 14 of the capture chamber 10 of the device 1. Specifically,the system comprises a pump for the extracorporeal circulation 30, aninlet valve 32 and outlet valve 34 for the entry into and the exit fromthe capture chamber 10, two manifolds 34 at the inlet 12 and the outletof the capture chamber 10, an air detector 37 downstream of the device 1and a detector for the inlet pressure 38 and a detector for the outletpressure 39. FIG. 6 shows, inside the system 100, a device 1 comprisinga capture element 20 with a helical reactant surface 22. However, thisis removable and replaceable by a different capture element 20″comprising a plurality of conical structures 22″.

FIG. 7 shows a detail of the system 100 with regard to the inlet 12 andthe outlet 14 of the capture chamber 10 of the device 1 according to analternative embodiment. In particular, the disclosed system is verysimilar to the one shown in FIG. 6 , the only difference being that thecapture element 20″′ consists of a helical structure followed by aplurality of conical structures. Therefore, the reactant surface 22″′ isdefined by a combination of conical and helical surfaces, thusincreasing the chances of capture of the cells to be removed by thedevice 1. Moreover, the combined presence of conical structuresincreases the clinical safety of the device 1 since it may blockpotential clots and emboli.

Lastly, FIG. 8 shows a block diagram describing the method 200 for theremoval of at least one biological and/or chemical entity C from anextracorporeal blood volume (B) according to the present invention.

The method 200 comprises the step of introducing 210 the extracorporealblood B into the capture chamber 10 of the device 1 having in itsinterior at least one capture element 20; 20′; 20″. Subsequently, themethod comprises the step of screening 220 the extracorporeal blood B bycontact with the reactant surface 22; 22′; 22″ of the capture element20; 20′; 20″ comprising a plurality of binding agents A for thebiological and/or chemical entity C to be removed. Finally, the methodcomprises the step of extracting 230 the extracorporeal blood B devoidof said biological and/or chemical entity C from the capture chamber 10.

The steps 210, 220 and 230 may be carried out after analyzing apredetermined volume of blood B. Above this value of blood volume B, themethod 200 comprises the step of stopping 240 the introduction ofextracorporeal blood B into the capture chamber 10 and removing thecapture element 20; 20′; 20″ to analyze the nature and quantity of thebiological and/or chemical entity C removed and present on the reactantsurface 22; 22′; 22″. Finally, in a subsequent step 250, the captureelement 20; 20′; 20″, once cleared of the entities previously captured,can be reintroduced into the capture chamber 10 and the method 200 canbe restarted from step 210. It should be noted that on the basis of theanalysis of the entities captured in step 240, the capture element 20;20′; 20″ may be replaced with a different one in terms of shape and/orconfiguration of the reactant surfaces 22; 22′; 22″ or in terms ofquantity, nature and distribution of the binding agents A.

A person skilled in the art, in order to meet further and contingentrequirements, may effect several further modifications and variations tothe device, the system and the method described above, all howevercomprised within the scope of protection of the present invention asdefined by the appended claims.

The invention claimed is:
 1. A method for the ex-vivo removal of abiological and/or chemical entity (C) from an extracorporeal bloodvolume (B) comprising: a) introducing the extracorporeal blood (B) intoa hollow capture chamber having a cylindrical shape, an inlet for theentry of the extracorporeal blood (B), an outlet for the outflow of theextracorporeal blood (B) and a capture element extending longitudinallywithin the capture chamber and movable relative to the capture chamber,the capture element: i) being rotatable about a longitudinal axis (a) ofthe capture chamber; ii) having a reactant surface for contacting theextracorporeal blood (B), said reactant surface comprising a pluralityof binding agents (A) for the biological and/or chemical entity to beremoved (C), and iii) a coupling and uncoupling system configured suchthat the capture element can be removed from the capture chamber toallow analysis of entities captured by the reactant surface, b)filtering the extracorporeal blood (B) by contact with the reactantsurface having the plurality of binding agents (A) for the biologicaland/or chemical entity (C) to be removed; and c) extracting theextracorporeal blood (B) devoid of said biological and/or chemicalentity (C) from the capture chamber; wherein the total volume ofextracorporeal blood (B) flowing inside the capture chamber is between 3to 30 liters of blood.
 2. The method according to claim 1, furthercomprising: d) stopping the introduction of extracorporeal blood (B)into the capture chamber after introducing a predetermined volume ofblood (B) and removing the capture element to analyze the nature andquantity of the biological and/or chemical entity (C) removed andpresent on the reactant surface.
 3. The method according to claim 1,further comprising diagnosing and/or monitoring the progression of atumor disease present in the extracorporeal blood wherein theextracorporeal blood is obtained from a patient suspected of havingcirculating tumor cell.
 4. The method according to claim 1, wherein thebiological and/or chemical entity to be removed (C) is a circulatingtumor cell.
 5. The method according to claim 1, wherein the bindingagents (A) comprise one or more antibodies, or adhesion proteins, oraptamers, thereby providing the removal of epithelial, mesenchymal,hybrid epithelial-mesenchymal, and stem-like CTCs (Circulating TumorCells), singly and/or in clusters.
 6. The method according to claim 1,wherein the reactant surface of the movable capture element is shapedlike a helix.
 7. The method according to claim 1, wherein the hollowcapture chamber further comprises one or more conical structures havingeach a filtering mesh surface made of holes with a diameter equal orgreater than 100 μm.
 8. The method according to claim 1, wherein theplurality of binding agents (A) is distributed evenly over the entirereactant surface of the capture element.
 9. The method according toclaim 1, wherein the plurality of binding agents (A) consists of bindingagents of a different nature, which are concentrated in specific areasof the reactant surface of the movable capture element.
 10. The methodaccording to claim 1, wherein the binding agents (A) are evenlydistributed or concentrated in specific areas on an inner surface of thecapture chamber.
 11. The method according to claim 1, further comprisingintroducing an anticoagulant into the extra corporeal blood prior to theextracorporeal blood being introduced into the hollow capture chamber.